The Roundabout family of receptors are molecular guidance molecules which regulate axon guidance, neuronal migration and leukocyte chemotaxis in response to interaction with Slit proteins (Suchting et al., FASEB J. 19:121-123 (2005)). Roundabout receptor molecules contain five immunoglobulin and three fibronectin domains in their extracellular region. Magic Roundabout (i.e., Robo4 or endothelial cell-specific molecule 4 (ESCSM4)) is structurally distinct from other roundabout family members. Human Magic Roundabout (Robo4) comprises two immunoglobulin and major histocompatibility complex domains (amino acids 46-116 and 151-209), two fibronectin type III domains (amino acids 252-335 and 347-432), a transmembrane region (468-490), and a proline rich region (amino acids 715-772) (see Huminiecki et al., Genomics 79(4):547-552 (2002), and FIG. 1 therein). Mouse and human Robo4 show 75% nucleotide sequence identity (Huminiecki et al., supra (2002)).
Robo4 expression analysis indicated that Robo4 expression is highly restricted, with strong expression in placenta and tumors including brain, bladder, and colonic metastasis to the liver, where tumor expression is restricted to tumor vasculature (Huminiecki et al., supra (2002)). In addition, Robo4 expression is associated with sites of active angiogenesis, but is not detected in neuronal tissue (Huminiecki, L. et al., supra (2002)).
As a membrane associated receptor with extracellular domains, Robo4 is a useful target for delivering cytotoxic therapeutics for inhibition of vascular endothelial cell proliferation during angiogenesis. Robo4 is also a useful target to delivery of detectable markers to proliferating vascular endothelial cells. Antibody conjugates targeted to Robo4 (also referred to as ECSM4) have also been reported as potential therapeutic compounds where the conjugate comprises a cytotoxin and as potential diagnostic markers where the conjugate comprises a detectable label (see, for example, WO 2002036771).
The use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Res. 19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drg Del. Rev. 26:151-172 (1997); U.S. Pat. No. 4,975,278) allows targeted delivery of the drug moiety to tumors, and intracellular accumulation of the drugs.
A number of antibody-drug conjugates that target other molecules have been or are being developed. For example, ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope conjugate composed of a murine IgG1 kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and 111In or 90Y radioisotope bound by a thiourea linker-chelator (Wiseman et al., Eur. Jour. Nucl. Med. 27(7):766-77 (2000); Wiseman et al. Blood 99(12):4336-42 (2002); Witzig et al., J. Clin. Oncol. 20(10):2453-63 (2002); Witzig et al., J. Clin. Oncol. 20(15):3262-69 (2002)). Although ZEVALIN has activity against B-cell non-Hodgkin's Lymphoma (NHL), administration results in severe and prolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), composed of a huCD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine (Immunogen, Inc.), composed of a huC242 antibody linked via the disulfide linker SPP to the maytansinoid drug moiety, DM1, is being developed for the treatment of cancers that express CanAg antigen, such as colon, pancreatic, gastric, and others. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), composed of an anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug moiety, DM1, is being developed for the potential treatment of prostate tumors. The same maytansinoid drug moiety, DM1, was linked through a non-disulfide noncleavable linker, SMCC, to a mouse murine monoclonal antibody, TA.1 (Chari et al., Cancer Res. 52:127-131 (1992)). This conjugate was reported to be 200-fold less potent than the corresponding disulfide linker conjugate.
The auristatin peptides, auristain E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin, have been conjugated to: (i) cBR96, a chimeric monoclonal antibody specific for Lewis Y on carcinomas; (ii) cAC10 which is specific to CD30 on hematological malignancies (Klussman et al., Bioconjugate Chemistry 15(4):765-773 (2004); Doronina et al. Nature Biotech. 21(7):778-784 (2003); Francisco et al. Blood 102(4):1458-1465 (2003); U.S. Patent Publication No. 2004/0018194; (iii) anti-CD20 antibodies such as Rituxan® (rituximab) (WO 04/032828) for the treatment of CD20-expressing cancers and immune disorders; (iv) anti-EphB2 antibodies 2H9 and anti-IL-8 for treatment of colorectal cancer (Mao et al. Cancer Res. 64(3):781-788 (2004)); (v) E-selectin antibody (Bhaskar et al. Cancer Res. 63:6387-6394 (2003)); and (vi) other anti-CD30 antibodies (WO 03/043583). Monomethylauristatin (MMAE) has also been conjugated to 2H9, an antibody against EphB2R which is a type 1 TM tyrosine kinase receptor with close homology between mouse and human, and is over-expressed in colorectal cancer cells (Mao et al., Cancer Res. 64:781-788 (2004)).
Monomethylauristatin MMAF, a variant of auristatin E (MMAE) with a phenylalanine at the C-terminus (U.S. Pat. Nos. 5,767,237 and 6,124,431), has been reported to be less potent than MMAE, but more potent when conjugated to monoclonal antibodies (Senter et al, Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented Mar. 28, 2004). Auristatin F phenylene diamine (AFP); a phenylalanine variant of MMAE was linked to an anti-CD70 mAb, 1F6, through the C-terminus of 1F6 via a phenylene diamine spacer (Law et al, Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 625, presented Mar. 28, 2004).
There exists a need in the art for additional drugs to treat diseases and disorders associated angiogenesis including, e.g., aberrant angiogenesis associated with cancers dependent on the growth and proliferation of vasculature of endothelial origin. There also exists a need in the art for additional endothelial cell-targeted anti-Robo4 antibody-drug conjugates for the detection and visualization of blood vessel growth and proliferation in, for example, cancers or ocular disorders supported or caused by excess proliferation of blood vessels, as well as in disorders or other physiological states in which monitoring blood vessel growth is useful in understanding or treating the physiological state. The present invention meets these and other needs.